WO2017141508A1

WO2017141508A1 - Platelet aggregation activity analysis device, platelet aggregation activity analysis system, platelet aggregation activity analysis program, and platelet aggregation activity analysis method

Info

Publication number: WO2017141508A1
Application number: PCT/JP2016/083896
Authority: WO
Inventors: 彩村田; 義人林; 賢三町田; マルクオレルブルン
Original assignee: ソニー株式会社
Priority date: 2016-02-17
Filing date: 2016-11-16
Publication date: 2017-08-24
Also published as: EP3418748A1; JP2021036246A; CN108603888B; JP7070641B2; JPWO2017141508A1; JP6941061B2; CN108603888A; US20190049428A1; EP3418748A4

Abstract

Provided are a novel platelet aggregation activity measurement device and a novel platelet aggregation activity measurement method, which can be used for monitoring in an anti-platelet therapy. A platelet aggregation activity analysis device equipped with: a blood coagulation system analysis section for analyzing platelet aggregation activity on the basis of measurement data for a coagulation process of a platelet-containing sample to which a platelet-inducing substance has been added; and an output control section for controlling the output of an analysis result produced by the blood coagulation system analysis section.

Description

Platelet aggregation ability analyzer, platelet aggregation ability analysis system, platelet aggregation ability analysis program, and platelet aggregation ability analysis method

The present invention relates to a platelet aggregation analysis device, a platelet aggregation analysis system, a platelet aggregation analysis program, and a platelet aggregation analysis method.

Conventional anticoagulant therapy and antiplatelet therapy are indispensable for the prevention of thrombosis, and their usefulness has been shown by large-scale clinical trials. However, the effect of antiplatelet therapy to reduce arterial thrombosis is lower than that of anticoagulant therapy to reduce cerebral infarction. In anticoagulant therapy, the efficacy of each anti-platelet drug can be sustained while the drug efficacy is appropriately monitored for each patient by the International Standard Ratio of Prothrombin Time (PT-INR) and thrombotest. One possible cause is the lack of established monitoring methods for antiplatelet therapy despite the reported differences. Therefore, even in antiplatelet therapy, if the drug efficacy can be appropriately monitored by an easy-to-understand method, it will be a means for searching for an appropriate method of using the drug in each case, and an improvement in the therapeutic effect can be expected.

The most basic functions of platelets are adhesion and aggregation. Platelet aggregation ability, which determines the adhesion state between platelets, is the most popular method for quantitatively measuring this basic phenomenon. The test methods include (1) permeability method and (2) impedance method. (3) There are particle counting methods. Depending on the type and concentration of the aggregation-inducing substance used in these test methods, it is possible to know details of the decrease and enhancement of the blood microscopic test function. In particular, the permeability method is common for daily inspection.

In the permeability method of (1), platelet aggregation is added and platelet aggregation is quantified over time using the increase in transparency of platelet-rich plasma accompanying platelet aggregation in platelet-rich plasma (PRP). Is the method.
Problems include the following (i) to (iv):
(i) Since the separation of plasma is essential, the sample processing up to the measurement is complicated, the amount of PRP obtained varies depending on the centrifuge conditions, and the platelet recovery rate is not constant. Also, during the PRP separation operation, high-density platelets precipitate together with erythrocytes, and their aggregation attitude cannot be observed.
(ii) The strength of platelet aggregation (aggregation rate) depends to some extent on the number of platelets in PRP, so when the platelet count is 100,000 / μl or less, the difference in absorbance before and after aggregation is small and slightly changed. Cannot be detected,
(iii) Testing with turbid plasma such as whole blood and milk plasma is impossible,
(iv) The correlation between the formation of platelet aggregates and light transmittance is poor,
Etc.

The impedance method of (2) is a method of detecting platelet aggregation as a change in electrical resistance between electrodes, and the platelet aggregation ability in whole blood can be observed. You can observe the attitude.
Problems include the following (v) and (vi):
(v) The initial decrease in electrical resistance is due to the presence of red blood cells between the electrodes, making it difficult to determine the initial state of platelet aggregation,
(vi) The aggregation pattern is not stable compared to the permeability method,
Etc.

The particle calculation method (3) is a method for determining the degree of aggregation by calculating the number of platelet aggregates or the number of single platelets not involved in the formation of aggregates with a Coulter counter.
Problems include the following (vii) to (ix):
(vii) The procedure is complicated,
(viii) Cannot record changes over time,
(ix) Platelet lysis by aggregator is miscalculated as single platelet loss,
Etc.

By the way, in the blood coagulation system analysis, in recent years, a method for measuring the dielectric constant of the blood coagulation process has been devised as a method capable of simply and accurately evaluating blood coagulation measurement (Patent Document 1).
In this method, blood is filled in a capacitor-like sample portion composed of a pair of electrodes, and an alternating voltage is applied thereto to measure a change in dielectric constant accompanying the blood coagulation process. Here, the blood sample was collected from a vein using citric acid as an anticoagulant. By adding a calcium chloride aqueous solution just before the start of measurement, the anticoagulant action of citric acid was canceled and the blood coagulation reaction was performed. To advance. By analyzing the data thus obtained according to a predetermined algorithm, parameters relating to blood coagulation such as blood coagulation time can be obtained.

The blood coagulation system analysis system apparatus based on the dielectric constant measurement can also acquire information on the influence of platelets on coagulation.
For example, by adding a substance that activates or inactivates platelets to blood, information on the coagulation ability of the blood is obtained based on a change that occurs in a complex dielectric constant spectrum measured during the blood coagulation process. System analysis can be performed (Patent Document 2). In this blood coagulation system analysis method, when a platelet activating substance is used as the substance, it is in an inactive state in the blood based on a change that occurs in the complex dielectric constant spectrum due to platelet activation by the substance. Information on the coagulation ability of the contained platelets can be acquired. In addition, when a platelet inactivating substance is used as the substance, information on the coagulation of platelets contained in the active state in the blood based on the change that occurs in the complex permittivity spectrum due to the platelet inactivation by the substance. Can be obtained.

On the other hand, in conventional blood coagulation tests such as International Standard Ratio of Prothrombin Time (PT-INR) and Activated Partial Thromboplastin Time (APTT), bleeding due to decreased blood coagulation ability due to excessive administration of anticoagulants Only risk can be evaluated, and the risk of thrombosis associated with increased blood coagulation ability cannot be evaluated. In addition, the existing platelet agglutination test using platelet-rich plasma (PRP) requires a centrifugation procedure, and platelets are activated during the procedure, so accurate test results cannot be obtained and the operation is complicated. It is.

In the conventional blood coagulation test, the following (x) and (xi):
(x) It is necessary to set a reference blood coagulation time reduction width Δt _s (reference value) in advance using a sample (whole blood) having normal coagulation ability.
(xi) When accelerating the coagulation reaction, the measurement time is long because no accelerating reagent is added (if the accelerating reagent is added, the difference due to platelet function becomes invisible)
There is a problem.

JP 2010-181400 A JP 2012-194087 A International Publication No. 2015/159623 Pamphlet

The inventors of the present application have conducted intensive research to provide a new method and apparatus for measuring platelet aggregation that can be used for monitoring in antiplatelet therapy, for example, and have completed the present technology.

In other words, this technology
A blood coagulation system analysis unit for analyzing platelet aggregation ability based on measurement data of a coagulation process of a platelet-containing sample to which a platelet-inducing substance is added;
There is provided a platelet aggregation analyzing apparatus including an output control unit for controlling an output of an analysis result by the blood coagulation system analysis unit.
The apparatus may include a biological sample holding unit that holds the platelet-containing sample and the platelet-inducing substance.
The apparatus may include a measuring unit that measures a coagulation process of the platelet-containing sample.
Furthermore, the apparatus may include a biological sample supply unit that supplies a platelet-containing sample.
Furthermore, the apparatus may include a drug supply unit that supplies the platelet-inducing substance.
The blood coagulation system analysis unit analyzes the measurement data of the coagulation process of the platelet-containing sample to which the platelet-inducing substance is added based on the measurement data of the coagulation process of the platelet-containing sample to which the platelet-inducing substance is not added. be able to.
The measurement information obtained by measuring the coagulation process of the platelet-containing sample may be measurement data of electrical characteristics and / or viscoelasticity.
The measurement data of the electrical characteristics may be a dielectric constant of a platelet-containing sample, and the measurement data of the viscoelasticity may be measurement data by a rheometer of the platelet-containing sample.
The platelet-containing sample is blood or plasma.
Furthermore, the blood or plasma may be collected from a subject who has been administered an antiplatelet aggregating agent or an anticoagulant.
The reaction between the platelet-containing sample and the platelet-inducing substance is performed for 1 minute to 20 minutes.
In addition, platelet aggregates can be collected after the reaction between the platelet-containing sample and the platelet-inducing substance.

This technology includes a blood coagulation system analysis unit that analyzes platelet aggregation ability based on measurement data of a coagulation process of a platelet-containing sample to which a platelet-inducing substance is added,
There is provided a platelet aggregation analysis system comprising a platelet aggregation analysis device including an output control unit for controlling the output of an analysis result by the blood coagulation system analysis unit, and a display device for displaying the analysis result.

The present technology includes a step of analyzing platelet aggregation ability based on measurement data of a coagulation process of a platelet-containing sample to which a platelet-inducing substance is added;
A program for analyzing platelet aggregation ability that causes a computer to execute a step of controlling the output of the analysis result is provided.

The present technology includes a step of analyzing platelet aggregation ability based on measurement data of a coagulation process of a platelet-containing sample to which a platelet-inducing substance is added;
And a method for analyzing the platelet aggregation ability, comprising the step of controlling the output of the analysis result.

It is a figure which shows the structure of a platelet aggregation ability analysis system. It is a figure which shows the structure of a platelet aggregation ability analyzer. It is a figure which shows the process of an analysis of platelet aggregation ability. It is a graph which shows the change of the platelet count by the platelet inducer addition to the blood. It is a graph which shows the measurement result of the whole blood by the platelet aggregation analysis apparatus using a dielectric constant. It is a graph which shows the measurement result of the whole blood by the platelet aggregation analysis apparatus using a dielectric constant. It is a graph which shows the measurement result of the whole blood by EXTEM of ROTEM. It is a graph which shows the measurement result of the whole blood by ROTEM INTEM. It is a graph which shows the measurement result of the plasma by ROTEM EXTEM. It is a graph which shows the measurement result of the plasma by ROTEM INTEM. It is a graph which shows the change of the number of platelets by the reaction time after adding the platelet inducer to whole blood. It is a graph which shows the measurement result of the whole blood by the platelet aggregation analysis apparatus using a dielectric constant. It is a graph which shows the measurement result of the whole blood by EXTEM of ROTEM. It is a graph which shows the measurement result of the whole blood by the platelet aggregation analysis apparatus using a dielectric constant. It is a graph which shows the measurement result of the whole blood by EXTEM of ROTEM. It is a graph which shows the effect of a filter. It is a graph which shows the measurement result of the whole blood which made the maximum value 1 by the platelet aggregation analysis apparatus using a dielectric constant. It is a graph which shows what computed the reciprocal number of the difference of the measurement result of the whole blood by the platelet aggregation analysis apparatus using a dielectric constant.

Hereinafter, preferred embodiments for carrying out the present technology will be described. In addition, embodiment described below shows typical embodiment of this technique, and, thereby, the range of this technique is not interpreted narrowly. The description will be given in the following order.
1. 1. Platelet aggregation ability analysis system Platelet aggregation ability analyzer 2-1. Configuration of apparatus 2-2. 2. Device operation Example 3-1. Outline of the present embodiment 3-2. Confirmation of platelet aggregation 3-3. When the specimen is whole blood 3-4. When the specimen is plasma 3-5. Effect of time for reacting platelets and inducer 3-6. When platelets are stimulated for a long time 3-7. Collection of aggregated platelets using a filter 3-8. Method for analyzing platelet aggregation 3-9. Summary

1. Platelet aggregation ability analysis system The platelet aggregation ability analysis system according to this technology is
A blood coagulation system analysis unit for analyzing platelet aggregation ability based on measurement data of a coagulation process of a platelet-containing sample to which a platelet-inducing substance is added;
A platelet aggregating ability analysis device including an output control unit for controlling an output of an analysis result by the blood coagulation system analysis unit; and a display device for displaying the analysis result.

FIG. 1 shows the configuration of a platelet aggregation ability analysis system.
The platelet aggregation ability analysis system 2000 includes a platelet aggregation ability analyzer 1000 and a display device 1010.
The platelet aggregation ability analysis system 2000 measures the platelet aggregation ability of a platelet-containing sample and analyzes the platelet aggregation ability from the measurement data.
The display device 1010 displays the analysis result of platelet aggregation ability.

The platelet aggregation analyzing apparatus 1000 may include a computer having a program for performing platelet aggregation based on measurement data, and the display device 1010 may be a display provided in the computer.

Further, the platelet aggregation analyzing apparatus 1000 may analyze not only the platelet aggregation ability of the platelet-containing sample but also other blood coagulation system measurement items. Examples of the other blood coagulation system measurement items include blood coagulation (clotting), fibrin formation, fibrin clot formation, clot formation, red blood cell formation, blood aggregation, red blood cell sedimentation (red sedimentation), blood clot Examples include contraction (retraction), hemolysis, and fibrinolysis. When analyzing these items, these item analysis programs are used in place of the platelet aggregation ability program provided in the computer.

Display device 1010 may include a warning unit. The warning unit presets a range of normal values for each blood state change, and issues a warning when the analysis result of the sample is outside the range of normal values.
The warning method is not particularly limited. For example, the warning can be given by display display or voice.

2. Platelet aggregation ability analyzer 2-1. Configuration of Apparatus FIG. 2 shows a detailed configuration of an example of the platelet aggregability analyzing apparatus 1000 described in FIG. The platelet agglutination analysis device 1000 includes a blood coagulation system analysis unit 12 and an output control unit 14, and more specifically, as main parts, a biological sample supply unit 2, a drug supply unit 3a, a reagent supply unit 3b, The biological sample holding units 1a and 1b and the measurement unit 10 are also included.

The biological sample supply unit 2 supplies blood collected from a human or a mammal to the platelet aggregation analyzing apparatus 1000.
The blood supply method is not particularly limited, and it is supplied from a syringe, puts blood in a sample cartridge and is set in the platelet aggregation analysis device 1000, or is directly supplied from the subject to the platelet aggregation analysis device 1000 through a tube. be able to. The subject may have been administered an antiplatelet aggregating agent or an anticoagulant.

The biological sample standby unit 4 enables the blood sent from the biological sample supply unit 2 to be maintained at a constant temperature and state. The biological sample standby unit 4 includes a temperature control unit 5 that maintains the temperature of the blood, a time control unit 6 that controls the standby time of the blood, and more preferably includes a stirring mechanism 7a.

The first biological sample holder 1a supplies the aforementioned platelet-inducing substance to blood and performs a reaction. A drug supply unit 3a is connected to the first biological sample holding unit 1a.
The drug supply unit 3a supplies platelet-inducing substances to the blood.
The first biological sample holding unit 1a includes a temperature control unit 5, a time control unit 6, and a stirring mechanism 7b. In the first biological sample holding unit 1a, stirring is performed while maintaining the temperature of the blood sent from the biological sample standby unit 4, and platelets in the blood and the platelet-inducing substance from the drug supply unit 3a are mixed for a certain period of time. React.

The biological sample sorting unit 8 sorts blood reacted with the platelet-inducing substance in the first biological sample holding unit. The collected blood is sent from the biological sample collection unit 8 to the second biological sample holding unit 1b.

The second biological sample holding part 1b supplies a coagulation promoting substance to blood and performs a reaction. A reagent supply unit 3b is connected to the second biological sample holding unit 1b.
The reagent supply unit 3b supplies a coagulation promoting substance to blood.
Examples of the coagulation promoting substance include calcium, an extrinsic coagulation promoting reagent, an intrinsic coagulation promoting substance, and the like. From these, one type or two or more types may be combined to promote coagulation. Specific exogenous coagulation promoting reagents include, for example, tissue thromboplastin, tissue factor and the like, and intrinsic coagulation promoting reagents include, for example, ellagic acid and kaolin.

Further, the second biological sample holding unit 1b includes a temperature control unit 5 and a stirring mechanism 7c, and may further include a drive mechanism 9. The drive mechanism 9 performs operations related to the second biological sample holding unit 1b, such as driving the temperature control unit 5 and the stirring mechanism 7c, and sending blood.
In the second biological sample holding unit 1b, the blood is reacted with the coagulation promoting substance from the reagent supply unit 3b while maintaining the blood at a constant temperature.

The measuring unit 10 measures the dielectric constant after or simultaneously with the addition of a coagulation promoting substance to blood. A rheometer may be used for the measurement unit 10. Examples of the rheometer include rotation thromboelastometry, thromboelastography, and Leo Rocks (ReoRox ™).

Measurement condition control unit 13 adjusts conditions suitable for each measurement method, such as temperature and measurement time. When the dielectric constant is measured by the measurement unit 10, the measurement condition control unit 13 controls, for example, the frequency used for measurement, the measurement interval, and the like.

In addition, the accuracy management unit 11 manages the data so as not to cause a difference between measurements, background fluctuations, etc., monitors the state of each part of the apparatus, etc., and includes this in the measurement unit 10. it can.
In addition, a platelet collection unit including a filter or the like for collecting aggregated platelets may be provided in front of the measurement unit 10.

Blood coagulation system analysis unit 12 detects and analyzes changes in blood state based on measurement data based on dielectric constant. The blood coagulation system analysis unit 12 outputs the analysis result to the output control unit 14. The output control unit 14 outputs and displays the analysis result on the display device 1010. In addition, the blood coagulation system analysis unit 12 communicates with the storage unit 15, and the storage unit 15 stores data and analysis results over time in continuous measurement of platelet-containing samples, or previously measured data and analysis results, etc. .

The blood aggregating ability analysis apparatus 1000 described above operates various logical functions of the platelet aggregating ability analyzing apparatus 1000 by a processor executing a program stored in a memory or another recording medium.

2-2. Operation of the apparatus First, blood is collected from a human or mammal. Whole blood may be used as a sample as it is, or blood may be stretched at 1000 rpm for 10 minutes, for example, and platelet plasma may be collected and used as a sample.

FIG. 3 shows a process of analyzing platelet aggregation ability.
The sample is first supplied to the biological sample supply unit 2. The sample is sent from the biological sample supply unit 2 to the biological sample standby unit 4, heated to 37 ° C., and stirred for 3 minutes, for example, and maintained (S1101). Next, the sample is sent to the biological sample holding unit 10, physiological saline is added to the control, and a platelet-inducing substance is added to the sample to be measured (S1102). The platelet-inducing substance to be added is not particularly limited, but collagen, epinephrine, ristocetin, calcium ion, thrombin, thromboxane A ₂ , thrombin receptor activating protein (TRAP), adenosine diphosphate (ADP), arachidonic acid (AA) Serotonin, adrenaline, noradrenaline and the like are used.

The concentration of the platelet-inducing substance is preferably 1 μM or more and 100 μM or less, more preferably 3 μM or more and 80 μM or less, and further preferably 5.4 μM or more and 62 μM or less for thrombin receptor activating protein (TRAP). In the case of adenosine diphosphate (ADP), it is preferably 0.1 μM or more and 100 μM or less, more preferably 0.5 μM or more and 30 μM or less, and further preferably 1.0 μM or more and 12.5 μM or less. In the case of arachidonic acid (AA), it is preferably 0.01 mM or more and 10 mM or less, more preferably 0.05 mM or more and 5.0 mM or less, and further preferably 0.08 mM or more and 1.0 mM or less.

After the physiological saline or platelet-inducing substance is added to the sample in the first biological sample holding unit 1a, the sample is continuously heated to 37 ° C. in the first biological sample holding unit 1a. Thereafter, stirring is preferably performed for 3 to 6 minutes (S1103), and platelets are consumed. When stirring for 1 minute or longer, the reaction between the platelets and the platelet-inducing substance proceeds. When stirring for 20 minutes or shorter, the platelet aggregates may not collapse.

Next, it is determined whether the collected (aggregated) platelets are collected (S1104). The collection can be performed, for example, by filtration (S1105).
The sample from which the aggregated platelets are determined to be collected and from which the aggregated platelets have been removed is subjected to extrinsic / intrinsic coagulation measurement by the measurement unit 10 provided in the second biological sample holding unit 1b (S1106).

Next, the blood coagulation system analysis unit 12 performs analysis according to the following procedure from the data of external / intrinsic system coagulation measurement.
First, the blood coagulation system analysis unit 12 extracts feature points based on the measurement result (S1107).
The characteristic points are, for example, the point where the measurement results of the control and the sample start to stabilize, the point where the clotting time of platelets is determined, and the like.
Next, the blood coagulation system analysis unit 12 calculates the difference between the control and the sample at the extracted feature points (S1108), and outputs the result.
The blood coagulation system analysis unit 12 determines that platelets are activated as the difference between the characteristic points of the control and the sample increases (S1109).
Next, the blood coagulation system analysis unit 12 outputs the analysis result to the display control unit 14, and the display control unit outputs and displays the analysis result on the display device 1010 (S1112).

Alternatively, when it is determined that the aggregated platelets are not collected, the extrinsic system / intrinsic system coagulation measurement is performed by the measurement unit provided in the second biological sample holding unit without collecting the aggregated platelets (S1110). .
Next, the contribution of platelets to coagulation is analyzed (S1111). The analysis can be performed by comparing the measurement result of the control added with the physiological saline and the measurement result of the sample added with the platelet-inducing substance.
For example, the measurement data obtained over time can be graphed, and the contribution to platelet coagulation can be analyzed by looking at the difference or ratio of the measured values at a specific time, the difference or ratio of the graph area, etc. . When the difference between the measured sample and the control becomes small, it is presumed that the platelet aggregation function is lowered or a platelet inhibitor is being taken.
Next, the blood coagulation system analysis unit 12 outputs the analysis result to the display control unit 14, and the display control unit outputs and displays the analysis result on the display device 1010 (S1113).

It should be noted that the procedure shown in the flowchart shown in FIG. 3 can be performed in parallel or individually even if not necessarily time-sequentially processed, as well as processing performed time-sequentially in the order described. Also includes processing to be performed. Further, it goes without saying that the order of the procedures processed in time series can be changed as appropriate in some cases.

In order to perform the above procedure, it is also possible to create a computer program incorporated in the hardware built in the platelet aggregation analyzing apparatus 1000 of the present technology. A storage medium storing the computer program is also provided by the present technology.

The above description mainly relates to a technique (see Patent No. 5691168, Patent No. 5678422) using a measurement method of a solidification process using electrical characteristics (dielectric constant). The coagulation process can also be measured by other methods.
Other methods include, for example, measurement of a coagulation process by viscoelasticity, and this method can also be applied to the present technology.

For the measurement of viscoelasticity, the aforementioned thromboelastography (TEG (registered trademark)) blood coagulation analyzer (Hemonetics), rotation thromboelastometry (ROTEM (registered trademark)) blood coagulation analyzer (Nippon Fingal Link) Etc. can be used.

In thromboelastography, a whole blood sample is injected into a cup, which is a measurement container, and an inducer according to the purpose of the test is added. Then, a rod-like pin suspended by a wire is immersed from the top of the container. Giving a steady reciprocating angular motion (typically reciprocating 4.45 ° in 10 seconds). As the coagulation reaction proceeds, the viscoelasticity of the specimen increases, the relative movement of the cup and pin decreases, and therefore the rotational displacement of the pin increases. By recording this rotational displacement with time using an optical system in the apparatus, a waveform called a thromboelastogram is obtained.

Rotation thromboelastometry is basically based on the same principle, although there is a difference that reciprocal angular motion is given to the pin instead of the cup. Whereas the prothrombin time and activated partial thromboplastin time are methods for detecting the end point of coagulation, thromboelastography and thromboelastometry are a series of processes from the start of coagulation to thrombus formation and subsequent fibrinolysis. There is an advantage that it can be monitored over time with a single device.

When measuring the coagulation process by the rotation thromboelastometry, both whole blood and plasma can be used as a platelet-containing sample. When the platelet aggregability analyzer 1000 is used, it is preferable to use whole blood.

3. Examples Hereinafter, examples of the examples will be described, but the present technology is not limited thereto.
3-1. Overview of this Example First, a platelet-inducing substance was added to whole blood or platelet-rich plasma (PRP). When stirring for a certain period of time, platelets showed an agglutination reaction, and the degree of the reaction was confirmed by a decrease in the number of platelets. The platelet count was measured by a multi-item automatic blood cell counter pocH-100i (Sysmex) based on the DC detection method that classifies the type according to the cell size.

3-2. Confirmation of platelet aggregation <Experimental method>
Venous blood collection of healthy subjects was performed by a normal method using a commercially available vacuum blood collection tube (blood collection amount: 1.8 mL, 5 tubes) containing citric acid as an anticoagulant. The first one was discarded without being used, and the remaining four were used for the experiment shown below. Further, after blood collection, it was used after being allowed to stand at room temperature for about 30 minutes.
First, 300 ul of whole blood was stirred for 3 minutes at 37 ° C. Thereafter, a platelet-inducing substance was added and allowed to react for 6 minutes. At this time, a system to which physiological saline was added was prepared as a control, and the number of platelets in the blood after the reaction was measured with a multi-item automatic blood cell counter pocH-100i (Sysmex).
In addition, in order to confirm reproducibility, a plurality of experiments were performed, and a similar experiment was also performed by a healthy person different from the above.

<Result>
FIG. 4 shows the blood platelet count as a control and the platelet count when a platelet-inducing substance is added to the blood. In FIG. 4, NaCl described as an additive reagent represents physiological saline and was used as a control. TRAP represents thrombin receptor activating protein, AA represents arachidonic acid, AA + ASA represents arachidonic acid and aspirin, ADP represents adenosine diphosphate, and ADP + PGE1 represents adenosine diphosphate and prostaglandin E1.

As shown in FIG. 4, it was found that the number of platelets was decreased by adding a platelet-inducing substance. From the results, it was found that platelet aggregation increased in the blood and single platelets decreased by the addition of platelet-inducing substances.

Substances that activate platelets include thrombin receptor activating protein (TRAP), adenosine diphosphate (ADP), arachidonic acid (AA), collagen, epinephrine, ristocetin, thromboxane A ₂ (TAX ₂ ), adrenaline, etc. In this example, TRAP, AA, and ADP were used.
The platelet count was also measured when substances that inactivate the respective activation pathways were added.
Thereby, when the inactivating substance was added, it turned out that even if an activator is added, a platelet aggregate does not increase and it exists as a single platelet.

3-3. When the sample is whole blood <Experimental method>
3-2. The blood coagulation ability was measured using the blood reacted in this experiment. This measurement uses the blood coagulation system analyzer (dielectric core gromometer prototype No. 2) that measures the dielectric constant of the sample and the comprehensive hemostasis measuring system ROTEM (registered trademark) at a measurement temperature of 37 ° C. went.
In the case of a blood coagulation system analyzer, DiCA-Ex is added with an aqueous calcium solution (0.2M Ca) mixed immediately before the start of measurement and a commercially available extrinsic stimulus (100-fold dilution + PBS (v / v = 1/5)). And DiCA-In to which a calcium aqueous solution (0.2MCa) and a commercially available endogenous stimulating reagent (+ PBS (v / v = 1/5)) were added were measured. The volume was unified at 12uL for each 180uL of blood.

In ROTEM, EXTEM using the principle of adding tissue thromboplastin or tissue factor as a coagulant, and INTEM using the principle of adding ellagic acid as an activator and adding partial thromboplastin were measured.
In addition, in order to confirm reproducibility, a plurality of experiments were performed, and a similar experiment was also performed by a healthy person different from the above.

<Result>
As shown in FIGS. 5A and 5B, the change in the dielectric constant (normalized) at 100 kHz over time is compared after adding physiological saline (control, + NaCl) and after adding platelet-inducing substance (+ TRAP). It was found that the amplitude increased when a platelet-inducing substance was added.
Also in ROTEM, as shown in FIG. 6-1 (EXTEM) and FIG. 6-2 (INTEM), a change in CF indicating clot hardness appeared.
This change was due to platelet consumption, and it was found that the platelet aggregation ability can be measured by ROTEM in the same way (FIGS. 6-1 and 6-2).

3-4. When the sample is plasma <Experimental method>
Venous blood collection for healthy individuals was performed by a conventional method using commercially available vacuum blood collection tubes (cigarette collection volume: 1.8 mL, 3 tubes) containing citric acid as an anticoagulant. The first one was discarded without being used, and the remaining two were used in the experiment shown below. In addition, after blood collection, the mixture was allowed to stand at room temperature for about 30 minutes, and then the two were centrifuged at 1000 rpm for 10 minutes to collect platelet-rich plasma (PRP).

First, 300ul of plasma was stirred for 3 minutes at 37 ° C. Thereafter, 20ul of platelet-inducing substance was added and allowed to react for 6 minutes. At this time, a system to which physiological saline was added was prepared as a control, and the number of platelets in the blood after the reaction was measured with a multi-item automatic blood cell counter pocH-100i (Sysmex). The measurement of EXTEM and INTEM was performed by ROTEM. In ROTEM, plasma can also be measured as a specimen.

<Result>
As shown in FIGS. 7-1 and 7-2, as in the case of whole blood, since a result of a change in CF indicating clot hardness was obtained, plasma containing platelets and whole blood were used as measurement samples. It was shown that can be used.

3-5. Effect of reaction time of platelets and inducer <Experimental method>
Using whole blood (the blood collection method is the same as the experiment in 3-2 above), an experiment was conducted on the reaction time (heating / stirring time) after the addition of the platelet-inducing substance. After adding a platelet-inducing substance to blood, the change in the number of platelets after each reaction time (1 to 45 minutes) was measured with pocH-100i (Sysmex).

<Experimental result>
As shown in FIG. 8, it was found that the platelet count was minimized between 3 and 6 minutes, and thereafter the number recovered with time. Therefore, it is presumed that the platelet function is most activated 3 to 6 minutes after the addition of the platelet-inducing substance, and it can be judged that the influence of the platelet aggregation on the coagulation reaction is most easily observed after this reaction time.

Although not shown in FIG. 8, the platelet count in the case where the blood inducer used as the specimen was not added was 340,000 / ul.
In addition, since the lower limit of the normal platelet count is 50,000 / ul, it is assumed that the change in coagulation ability due to the change in platelet aggregation becomes difficult after 45 minutes after the addition of platelet-inducing substances. . This was confirmed in the experiment.

3-6. When platelets are stimulated for a long time <Experimental method>
Venous blood collection of healthy subjects was performed by a normal method using a commercially available vacuum blood collection tube (blood collection amount: 1.8 mL, 5 tubes) containing citric acid as an anticoagulant. The first one was discarded without being used, and the remaining four were used for the following experiments. Further, after blood collection, it was used after being allowed to stand at room temperature for about 30 minutes.

First, 300 ul of whole blood was stirred for 3 minutes at 37 ° C. Thereafter, 20ul of platelet-inducing substance was added and reacted for 6 minutes or 30 minutes. Using the reacted blood, coagulation ability was measured. The apparatus and reagent concentration used for the measurement are the same as those in 4-3. It was the same as the experiment.
At this time, a system to which the same amount of physiological saline as the platelet-inducing substance was added was also measured as a control.

<Result>
As shown in FIGS. 9-1 and 9-2, it was found that when the platelet-inducing substance was added and heated and stirred for 30 minutes, it approached the system to which no platelet-inducing substance was added. That is, said 4-5. The experimental results were confirmed.

3-7. Collection of aggregated platelets by filter etc. <Experimental method>
Similar to the above experimental method, platelets are activated by adding platelet-inducing substances to whole blood, then the reacted whole blood is passed through a filter (PluriStrainer), and DiCa-EX is analyzed with a blood coagulation system analyzer. EXTEM was measured with ROTEM.
The size of the filter used was 15 to 25 um, which is the size of a small aggregate of platelets. In this experiment, the result when 15um is used is shown.

<Result>
In the case of DiCa-Ex, as shown in FIG. 10-1, no significant difference was observed between the system through the filter and the system through the filter. That is, the system in which platelets exist as aggregates and the system removed by a filter show almost the same ratio, so the presence of aggregates is presumed to have no effect on the difference from the control. It was done.

On the other hand, in the case of ROTEM-EXTEM, as shown in FIG. 10-2, the effect of adding the inducer varies depending on whether or not the platelet aggregate is removed using a filter. When ROTEM was used, it was found that the difference from the control was better seen when the platelet aggregate was physically removed before the coagulation measurement.
Examples of other methods for removing platelet aggregates include a method using microbeads coated with an activated platelet-specific surface antibody before the start of measurement.

In FIG. 10-1 (DiCa-Ex) and FIG. 10-2 (ROTEM-EXTEM), the examination time is 15 minutes, which is determined by the coagulation time, and the value is 15 minutes (DiCa is the difference between the maximum value 1 and the 15-minute value) The ratio of NaCl-TRAP (without filter) and NaCl + Filter-TRAP + Filter (with filter) was calculated.
The results are shown in FIG.
In the case of DiCa-Ex, there was no significant difference in the ratio without the filter, but in the case of ROTEM, the difference in the ratio was greater with the filter.

3-8. Method for analyzing platelet aggregation ability 3-3. An example of the analysis method is shown based on the measurement result of the platelet coagulation analyzer based on the dielectric constant.
FIG. 12-2 shows a calculation time (calculation CF) obtained by calculating the reciprocal of the difference from 10 minutes determined as the examination time (FIG. 12-1), the maximum value being 1, and the difference from the maximum value. When NaCl was added (control), the average value was 17.7, and when the platelet-inducing substance was added, the average value was 12.7 (n = 4).

The range indicated by the arrow in FIG. 12-2 is reflected by the platelet function (platelet contributing part), and this difference may be reduced when the platelet aggregation function is lowered or when a platelet inhibitor is taken. is assumed. This was actually confirmed by a combination of ADP and a prostaglandin E1 inhibitor (ADP pathway inhibitor).

In the t-test, since p = 0.001 (p <0.01), it was judged that there was a significant difference between the two average values when NaCl was added and when the platelet-inducing substance was added.
(Here, in the t distribution, if it is in the outer 5% range, it is considered that there is a significant difference, not a sample from the same population, and the probability of being outside the 95% confidence interval is p.)

In addition to the above analysis method, a method of calculating the difference from the control by the area or ratio when the measurement data of the obtained coagulation process is graphed can be considered. Since the view of the blood coagulation system analyzer also changes depending on the measurement frequency, it has a higher degree of freedom than ROTEM, and it may be possible to calculate parameters other than platelet function at the same time.

In the present technology, the following steps are also performed:
(1) Taking measurement data of the coagulation process of a platelet-containing sample to which no platelet-inducing substance is added (data showing the aggregation effect of platelets and fibrin is obtained)
(2) On the other hand, measurement data of the coagulation process of a platelet-containing sample to which a platelet function inhibitor has been added is obtained (data obtained by excluding platelets (for fibrin) is obtained), and (3) Take measurement data of the coagulation process of the added platelet-containing sample,
The analysis method including this can be performed. When the subject is taking an antiplatelet drug, the measurement result appears between the data (1) and (2) depending on the degree of the effect.

3-9. Summary According to the present technology, the blood coagulation system analyzer can be used not only for the measurement of coagulation factors but also for the measurement of platelet function as a platelet aggregability analyzer, and can be used for monitoring treatment according to the type of antiplatelet drug. it can.
In addition, according to the present technology, it is possible to perform measurement with whole blood, which is a superior sample, as a clinical test with thrombosis in view.

In addition, this technique can also take the following structures;
[1] a blood coagulation system analysis unit for analyzing platelet aggregation ability based on measurement data of a coagulation process of a platelet-containing sample to which a platelet-inducing substance is added;
An apparatus for analyzing platelet aggregation, comprising an output control unit for controlling the output of the analysis result by the blood coagulation system analysis unit.
[2] The platelet aggregation analyzing apparatus according to [1], further comprising a biological sample holding unit that holds the platelet-containing sample and the platelet-inducing substance.
[3] The platelet aggregation analyzing apparatus according to [1] or [2], further including a measuring unit that measures a coagulation process of the platelet-containing sample.
[4] The platelet aggregation analyzing apparatus according to any one of [1] to [3], further comprising a biological sample supply unit that supplies the platelet-containing sample.
[5] The platelet aggregation analyzing apparatus according to any one of [1] to [4], further comprising a drug supply unit that supplies the platelet-inducing substance.
[6] The blood coagulation system analysis unit measures the coagulation process of the platelet-containing sample to which the platelet-inducing substance is added based on the measurement data of the coagulation process of the platelet-containing sample to which the platelet-inducing substance is not added. The apparatus for analyzing platelet aggregation ability according to any one of [1] to [5].
[7] The platelet aggregation analysis apparatus according to any one of [1] to [6], wherein the measurement information obtained by measuring the coagulation process of the platelet-containing sample is measurement data of electrical characteristics and / or viscoelasticity.
[8] The platelet aggregation analysis apparatus according to [7], wherein the measurement data of the electrical characteristics is a dielectric constant of a platelet-containing sample.
[9] The platelet aggregation analyzing apparatus according to [7], wherein the measurement data of the viscoelasticity is measurement data of a platelet-containing sample using a rheometer.
[10] The platelet aggregation analyzing apparatus according to any one of [1] to [9], wherein the platelet-containing sample is blood or plasma.
[11] The platelet aggregation analyzing apparatus according to [10], wherein the blood or plasma is collected from a subject who has been administered an antiplatelet aggregation agent or an anticoagulant.
[12] The platelet aggregation analyzing apparatus according to any one of [1] to [11], wherein the platelet-containing sample and the platelet-inducing substance are reacted for a time of 1 minute to 20 minutes.
[13] The platelet aggregation analysis apparatus according to [12], wherein platelet aggregation is collected after the reaction between the platelet-containing sample and the platelet-inducing substance.
[14] a blood coagulation system analysis unit for analyzing platelet aggregation ability based on measurement data of a coagulation process of a platelet-containing sample to which a platelet-inducing substance is added;
A platelet aggregation capacity analysis system comprising: a platelet aggregation capacity analysis apparatus including an output control section for controlling output of analysis results by the blood coagulation system analysis section; and a display device for displaying the analysis results.
[15] An analysis function for analyzing platelet aggregation ability based on measurement data of a coagulation process of a platelet-containing sample to which a platelet-inducing substance is added;
A program for analyzing platelet aggregation ability that causes a computer to execute an output control function for controlling the output of the analysis result.
[16] analyzing platelet aggregation ability based on measurement data of a coagulation process of a platelet-containing sample to which a platelet-inducing substance is added;
A method for analyzing platelet aggregation, comprising a step of controlling the output of the analysis result.

DESCRIPTION OF SYMBOLS 1a 1st biological sample holding part 1b 2nd biological sample holding part 2 Biological sample supply part 3a Drug supply part 3b Reagent supply part 4 Biological sample waiting part 5 Temperature control part 6 Time control part 7a, 7b Stirring mechanism 8 Biological sample Sorting unit 9 Drive mechanism 10 Measuring unit 11 Accuracy management unit 12 Blood coagulation system analysis unit 13 Measurement condition control unit 14 Output control unit 15 Storage unit 1000 Platelet aggregation analysis device 1010 Display device 2000 Platelet aggregation analysis system

Claims

A blood coagulation system analysis unit for analyzing platelet aggregation ability based on measurement data of a coagulation process of a platelet-containing sample to which a platelet-inducing substance is added;
An apparatus for analyzing platelet aggregation, comprising an output control unit for controlling the output of the analysis result by the blood coagulation system analysis unit.
The platelet aggregation analyzing apparatus according to claim 1, further comprising a biological sample holding unit for holding the platelet-containing sample and the platelet-inducing substance.
The platelet aggregation analyzing apparatus according to claim 1, further comprising a measuring unit for measuring a coagulation process of the platelet-containing sample.
The platelet aggregation analyzing apparatus according to claim 1, further comprising a biological sample supply unit that supplies the platelet-containing sample.
The platelet aggregation analyzing apparatus according to claim 1, further comprising a drug supply unit that supplies the platelet-inducing substance.
The blood coagulation system analysis unit analyzes the measurement data of the coagulation process of the platelet-containing sample to which the platelet-inducing substance is added based on the measurement data of the coagulation process of the platelet-containing sample to which the platelet-inducing substance is not added. The platelet aggregation analyzing apparatus according to claim 1.
The platelet aggregation analysis apparatus according to claim 1, wherein the measurement information obtained by measuring the coagulation process of the platelet-containing sample is measurement data of electrical characteristics and / or viscoelasticity.
The platelet aggregation analysis apparatus according to claim 7, wherein the measurement data of the electrical characteristics is a dielectric constant of a platelet-containing sample.
The platelet aggregation analysis apparatus according to claim 7, wherein the measurement data of the viscoelasticity is measurement data of a platelet-containing sample by a rheometer.
2. The platelet aggregation analyzing apparatus according to claim 1, wherein the platelet-containing sample is blood or plasma.
The blood platelet or plasma analysis apparatus according to claim 10, wherein the blood or plasma is collected from a subject who has been administered an antiplatelet aggregating agent or an anticoagulant.
The platelet aggregation analysis apparatus according to claim 1, wherein the platelet-containing sample and the platelet-inducing substance are reacted in a time of 1 minute to 20 minutes.
The platelet aggregation analyzing apparatus according to claim 12, wherein platelet aggregation is collected after the reaction between the platelet-containing sample and the platelet-inducing substance.
A blood coagulation system analysis unit for analyzing platelet aggregation ability based on measurement data of a coagulation process of a platelet-containing sample to which a platelet-inducing substance is added;
A platelet aggregation analysis system comprising: a platelet aggregation analysis device including an output control unit that controls output of an analysis result by the blood coagulation system analysis unit; and a display device that displays the analysis result.
Analyzing platelet aggregation ability based on measurement data of the coagulation process of a platelet-containing sample to which a platelet-inducing substance is added;
A program for analyzing platelet aggregation ability that causes a computer to execute a step of controlling the output of the analysis result.
Analyzing platelet aggregation ability based on measurement data of the coagulation process of a platelet-containing sample to which a platelet-inducing substance is added;
A method for analyzing platelet aggregation, comprising a step of controlling the output of the analysis result.